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Secretome analysis defines the major role of SecDF in Staphylococcus aureus virulence.

Quiblier C, Seidl K, Roschitzki B, Zinkernagel AS, Berger-Bächi B, Senn MM - PLoS ONE (2013)

Bottom Line: Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant.Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells.Virulence was significantly reduced using a Galleria mellonella insect model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

ABSTRACT
The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections.

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Adhesion, cytotoxicity and invasion in HUVECs.Interactions of Newman and the CHE482 strain set the wild type CHE482, CHE482 ΔsecDF and the complemented mutant CHE482 ΔsecDF pSecDF with HUVECs. (A) Effect of secDF inactivation in the CHE482 background on adhesion. (B) Growth in invasion medium. The arrow indicates the time point at which extracellular bacteria are lysed. (C) Inoculum dependent cytotoxicity of wild-type strains as determined by the MTT assay. (D) Effect of secDF inactivation in the CHE482 background on cytotoxicity as determined by the MTT assay. (E) Effect of secDF inactivation in the CHE482 background on invasion. ***, P<0.0001.
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pone-0063513-g004: Adhesion, cytotoxicity and invasion in HUVECs.Interactions of Newman and the CHE482 strain set the wild type CHE482, CHE482 ΔsecDF and the complemented mutant CHE482 ΔsecDF pSecDF with HUVECs. (A) Effect of secDF inactivation in the CHE482 background on adhesion. (B) Growth in invasion medium. The arrow indicates the time point at which extracellular bacteria are lysed. (C) Inoculum dependent cytotoxicity of wild-type strains as determined by the MTT assay. (D) Effect of secDF inactivation in the CHE482 background on cytotoxicity as determined by the MTT assay. (E) Effect of secDF inactivation in the CHE482 background on invasion. ***, P<0.0001.

Mentions: In a next step, we investigated whether the secDF deletion would also affect adherence to host endothelial cells (human umbilical vein endothelial cells, HUVECs). Because of the point mutation in the FnBPs of strain Newman mentioned above, this strain has been previously shown to be weakly adherent to HUVECs [56]. Therefore, we used the secDF mutant of strain CHE482 to test adherence and found significantly reduced adherence as compared to the wild type strain (Figure 4A). This effect was restored in the complemented mutant CHE482 ΔsecDF pSecDF. In accordance with the previous findings [56] adherence by strain Newman was only 12.7±6.6% of strain CH482.


Secretome analysis defines the major role of SecDF in Staphylococcus aureus virulence.

Quiblier C, Seidl K, Roschitzki B, Zinkernagel AS, Berger-Bächi B, Senn MM - PLoS ONE (2013)

Adhesion, cytotoxicity and invasion in HUVECs.Interactions of Newman and the CHE482 strain set the wild type CHE482, CHE482 ΔsecDF and the complemented mutant CHE482 ΔsecDF pSecDF with HUVECs. (A) Effect of secDF inactivation in the CHE482 background on adhesion. (B) Growth in invasion medium. The arrow indicates the time point at which extracellular bacteria are lysed. (C) Inoculum dependent cytotoxicity of wild-type strains as determined by the MTT assay. (D) Effect of secDF inactivation in the CHE482 background on cytotoxicity as determined by the MTT assay. (E) Effect of secDF inactivation in the CHE482 background on invasion. ***, P<0.0001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643904&req=5

pone-0063513-g004: Adhesion, cytotoxicity and invasion in HUVECs.Interactions of Newman and the CHE482 strain set the wild type CHE482, CHE482 ΔsecDF and the complemented mutant CHE482 ΔsecDF pSecDF with HUVECs. (A) Effect of secDF inactivation in the CHE482 background on adhesion. (B) Growth in invasion medium. The arrow indicates the time point at which extracellular bacteria are lysed. (C) Inoculum dependent cytotoxicity of wild-type strains as determined by the MTT assay. (D) Effect of secDF inactivation in the CHE482 background on cytotoxicity as determined by the MTT assay. (E) Effect of secDF inactivation in the CHE482 background on invasion. ***, P<0.0001.
Mentions: In a next step, we investigated whether the secDF deletion would also affect adherence to host endothelial cells (human umbilical vein endothelial cells, HUVECs). Because of the point mutation in the FnBPs of strain Newman mentioned above, this strain has been previously shown to be weakly adherent to HUVECs [56]. Therefore, we used the secDF mutant of strain CHE482 to test adherence and found significantly reduced adherence as compared to the wild type strain (Figure 4A). This effect was restored in the complemented mutant CHE482 ΔsecDF pSecDF. In accordance with the previous findings [56] adherence by strain Newman was only 12.7±6.6% of strain CH482.

Bottom Line: Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant.Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells.Virulence was significantly reduced using a Galleria mellonella insect model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

ABSTRACT
The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections.

Show MeSH
Related in: MedlinePlus